The Ras-related GTP-binding protein, Cdc42, is activated by a variety of extracellular stimuli and has been implicated in a number of fundamentally important cellular processes including the establishment of cell polarity and motility through influences on the actin cytoskeleton, and the regulation of cell-cycle progression through signaling pathways that lead to the nucleus. The tight regulation of the GTP-binding/GTPase cycle of Cdc42 is critical to its cellular functions, such that the accelerated cycling of Cdc42 between its GDP- and GTP-bound states leads to cellular transformation and tumor formation in nude mice. During the past funding period, we have combined biochemical, structural biology- and genetic approaches to study the regulation of the GTPbinding/GTPase cycle of Cdc42 by its guanine nucleotide exchange factor (GBF), the Dbl oncoprotein, and other regulatory proteins. We also have identified new potential regulators and target/effector molecules that may mediate its cellular actions, including the Dbl-related Cool (for cloned-out-of-a library) proteins and the tyrosine kinase, ACK-2 (for activated Cdc42 kinase-2). In this renewal application, we plan to extend this work to better understand two major aspects of Cdc42 function, namely the molecular basis by which Cdc42 and its signaling pathways are activated in cells (Aim l) and the molecular and cellular consequences of these activation events (Aim 2). This will constitute 4 lines of study. Aim 1a.) Studies of the Dbl gene product as a prototypical guanine nucleotide exchange factor for Cdc42. Here, the emphasis will be to determine how the guanine nucleotide exchange and cell growth-promoting activities of Dbl are regulated. Aim lb.) Understanding how a new family of Dbl-related (Cool) molecules is able to exhibit a diversity of regulatory effects on Cdc42/Rac- signaling through the p2l-activated kinase (PAK). Aim 2a.) The role of the newly discovered tyrosine kinase ACK-2 in Cdc42-signaling and its possible interplay with PAK. Aim 2b.) The role of IQGAP in Cdc42 signaling, with a particular emphasis on its potential involvement in Cdc42-mediated cell growth and transformation. The results of these studies should yield important insights into the molecular basis by which Cdc42 initiates a variety of signaling activities. They also should provide new clues toward understanding how cells, through the activation of Cdc42 and related GTP-binding proteins, coordinate a diversity of responses including morphological and actin cytoskeletal changes with the regulation of cell cycle progression and cytokinesis.